Pharmaceutical composition, use of mefloquine in a fixed dose, and method for treating tuberculosis

ABSTRACT

This invention concerns the use of mefloquine in relation to Mycobacterium tuberculosis. This invention also concerns the combination of mefloquine with drugs used in first and second choice treatment of tuberculosis, achieving a reduction in the treatment period of tuberculosis (TB) and the treatment of multi-drug resistant tuberculosis (MDR-TB).

FIELD OF INVENTION

This invention concerns, in its broadest conception, the use ofmefloquine (MFL) in relation to Mycobacterium tuberculosis.

BASES OF THE INVENTION

Tuberculosis (TB) is an ancient infectious disease caused byMycobacterium tuberculosis and continues to be the main cause of deathby infectious disease around the world. Mycobacterium tuberculosis wasdiscovered and identified in 1882 by Robert Koch and, in honor of him,it is also known as Koch's bacillus (BK).

The treatment of TB is based on a fixed combined dose of four drugs:rifampicin (RIF), isoniazid (INH), pyrazinamide (PYR) and ethambutol(ETB). The fixed dose regime is designed not only to prevent thesymptoms caused by active TB, but also to prevent the development ofresistant bacteria, frequently observed in the case of monotherapy.However, the period of treatment is long (six months), and theappearance of side effects in individuals is very common. As aconsequence of this, despite the fact that the current treatment hasreduced the number of deaths caused by TB, high levels of lapsing andinterruption have been observed. This fact has directly contributed tothe emergence of resistant strains of Mycobacterium tuberculosis.

Currently, the World Health Organization (WHO) classifies resistance toTB at two levels: multidrug-resistant tuberculosis (MDR-TB) andextensively drug-resistant tuberculosis (XDR-TB). MDR-TB is caused bybacteria that are resistant at least to INH and RIF. In the case ofXDR-TB, the bacteria are resistant to INH and RIF, as well as onefluoroquinolone and an injectable drug used in second-choice treatment(amikacin, kanamycin and capreomycin). These forms of TB do not respondto the standard six month treatment, its being necessary to use moretoxic and less effective drugs. As a result, in addition to beingprolonged (around two years), the treatment also becomes much morecostly.

With the appearance of HIV, tuberculosis chemotherapy has undergonegreat changes. The appearance of multi-drug resistant strains, above allthose resistant to rifampicin and isoniazid, has created many publichealth problems. As a result, it has been necessary to resort to newmedicaments which has led to the classification of anti-tuberculosisdrugs in two groups:

(1) the primary ones (first line or first choice) which are more potentand less toxic; and,(2) the secondary ones (second line or second choice) which are lesspotent and more toxic.

The drugs characterized as “first line” include isoniazid (INH),rifampicin (RIF), ethambutol (ETB) and pyrazinamide (PYR); and thesecond line drugs include streptomycin (SM) ethionamide (Et),aminosalicylic acid (Aa), cycloserine (Cs), amikacin (Am), kanamycin(Cn), capreomycin (Cp) and linezolid (L).

Mao et al., [ChemMedChem. 2007 November; 2(11):1624-30. Design,synthesis, and pharmacological evaluation of mefloquine-based ligands asnovel antituberculosis agents. Mao J, Wang Y, Wan B, Kozikowski A P,Franzblau S G.] mention that mefloquine analogues were developed andthat they were evaluated regarding anti-TB activity againstMycobacterium tuberculosis H37Rv.

Lowell S. Young et al. [Antimicrob. Agents Chemother. November 2009 vol.53 no. 11 4577-4579, Reconsidering Some Approved Antimicrobial Agentsfor Tuberculosis, Published ahead of print 8 Sep. 2009, doi:10.1128/AAC.00887-09] report that mefloquine, which is active againststrains resistant to cloroquine, possesses bactericidal activity againstMycobacterium Avium Complex (MAC), the most common infection bynontuberculosis mycobacteria. A single report of a human case describedthe successful treatment of a patient [rendered] resistant to MACdisease through the addition of linezolid and mefloquine to otheranti-Mac agents, though the first compound has limitations in long termtherapy. The article speculated that mefloquine also has an effectagainst M. tuberculosis and may be a substitute for isoniazid andrifampicin.

Raoni et al., [in Bioorganic & Medicinal Chemistry, Volume 20, Issue 1,1 Jan. 2012, Pages 243-248. Mefloquine-oxazolidine derivatives, derivedfrom mefloquine and arenecarbaldehydes: In vitro activity includingagainst the multidrug-resistant tuberculosis strain T113] present astudy where new mefloquine-oxazolidine derivatives show improvedanti-tuberculosis activity in relation to first line drugs.

It is worth highlighting that the studies of Mao at al. e Raoni at al.are reports of modifications made to the mefloquine molecule (newsubstances) which have still not undergone a series of tests such asanimal model and toxicological tests. Only in vitro tests have been doneand they were not tested with other drugs, which is to say they arebasic studies in the development of new drugs.

The study of Lowell S. Young et al. offers a good example of thepotential of mefloquine in combination with other drugs, in this caseusing linezolid. However, the author used only linezolid which has notyet been approved as an anti-TB drug.

In this context, the development of new, more efficient and less toxiccombinations which also contribute to reducing the treatment time ofdifferent forms of TB, with reduced side effects, is considered a globalpriority in the public health sphere.

SUMMARY OF THE INVENTION

This invention presents a wholly different and nonobvious concept inrelation to the other studies reported in the literature, which is acombination of different drugs used in the treatment of tuberculosiswith mefloquine demonstrating that this drug presents an importantsynergism when combined with other anti-TB drugs, presenting significantpotential in the development of a new combination (medicament) in thetreatment of resistant tuberculosis.

This invention concerns, in its broadest conception, the use ofmefloquine in relation to Mycobacterium tuberculosis to combattuberculosis.

This invention also concerns the use of mefloquine against Mycobacteriumtuberculosis combined with anti-tuberculosis agents for more effectivetreatment of tuberculosis.

This invention presents a composition for the treatment of tuberculosis.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the chemical structure of the drug mefloquine.

FIGS. 2A-2L show the chemical structures of some drugs used incombination with mefloquine.

DETAILED DESCRIPTION OF THE INVENTION

In the search for new treatments to combat tuberculosis (TB), the herebyinventors identified mefloquine (MFL) as being potentially useful in thetreatment of TB.

Among the significant aspects that were considered by the inventors inthe selection of mefloquine for use in a combined fixed dose formulationfor the treatment of tuberculosis, were: (i) its relatively longhalf-life; (ii) that it is well-tolerated by patients with HIV; (iii)that it did not present medical interactions with anti-retrovirals suchas protease inhibitors; (iv) that it presented good gastrointestinalabsorption; and, (v) that it concentrates mainly in the lungs, the organmost affected by M. tuberculosis.

MFL is currently used in malaria prophylaxis (World Malaria Report).However, MFL is also active against different species of Gram-positivebacteria (Kunin C M et al). Nevertheless, Fu L M et al. report that thephylogenetic position of Mycobacterium tuberculosis in relation to otherbacteria is controversial. The cell wall of Mycobacterium tuberculosishas characteristics of both Gram-positive and Gram-negative bacteria. Inthe genome tree constructed based on the conserved gene content, M.tuberculosis is more closely related to Gram-negative bacteria thanGram-positive bacteria.

Even though the state of the art is controversial, as described above,the inventors continued their search to understand the effect ofmefloquine in the treatment of tuberculosis, especially when combinedwith other drugs.

The inventors' search revealed a synergic effect of the combination ofmefloquine with first line and second line drugs used in the treatmentof tuberculosis.

Thus, this invention concerns the use of mefloquine in relation toMycobacterium tuberculosis.

The invention also encompasses the combination of mefloquine with drugsused in first and second choice treatment of tuberculosis, achieving apotential reduction in the treatment period of the tuberculosis (TB) andin the treatment of multi-drug resistant tuberculosis (MDR-TB).

It is also an objective of this invention to provide an effectivecombination containing mefloquine to treat tuberculosis that includesvarieties of Mycobacterium tuberculosis which are multiply resistant todrugs used in the treatment of tuberculosis.

This invention presents a pharmaceutical composition for treatingtuberculosis in a mammal that encompasses the administration, to saidmammal in need thereof, of an effective quantity of mefloquine incombination with agents effective in the treatment of tuberculosis.

The pharmaceutical preparations used in accordance with this inventionare prepared by mixing one or more agents effective in the treatment oftuberculosis with mefloquine.

The proportions used both of mefloquine and the drugs (agents useful inthe treatment of tuberculosis) used in the pharmaceutical composition ofthis invention vary from 0.01-10.

The number of drugs combined with mefloquine is a maximum of threedrugs.

This invention shall now be described with reference to the followingexamples which should not be interpreted as limiting the scope of theinvention.

Materials and Methods 1.1 Mycobacterium Tuberculosis

-   -   The following strains of M. tuberculosis were used:    -   standard strain M. tuberculosis H37Rv (ATCC 27294);    -   resistant strain M tuberculosis T3609, resistant to ofloxacin        (OFX) and streptomycin; and,    -   multi-resistant strain T113, resistant to isoniazid (INH),        rifampicin (RIF), ethambutol (ETB) and ofloxacin (OFX).

All the strains belong to the collection of the laboratory ofBiotechnology and Biotests of the Evandro Chagas Institute of ClinicalResearch (Bacteriologia e Bioensaio do Instituto de Pesquisa ClínicaEvandro Chagas—IPEC) of the Oswaldo Cruz Foundation.

1.2 Substances Used

In the tests carried out a minimum inhibitory concentration (MIC) ofmefloquine was used in relation to the strains of M. tuberculosispreviously mentioned here (item 1.1). Mefloquine was tested incombination with:

-   -   first choice drugs: isoniazid (INH), pyrazinamide (PYR),        rifampicin (RIF) and ethambutol (ETB).    -   second choice drugs in the treatment of tuberculosis.    -   linezolid (LYN).    -   fluoroquinolones: gatifloxacin (GAT), moxifloxacin (MCX),        sparfloxacin (SPR), ofloxacin (CFX), ciprofloxacin (CPX) and        levofloxacin (LVX).

1.3 Proportions and Number of Substances Used

The proportions used both of mefloquine and the drugs used varied from0.01-10. The number of drugs combined with mefloquine is a maximum ofthree.

1.3 Proportions and Numbers of Substances Used

The proportions used both of mefloquine and the drugs used varied from0.01-10 p/p. The number of drugs combined with mefloquine is a maximumof three.

Combination MIC_(combination) Proportion Mefloquine 12.5 μg/mL —Mefloquine + Linezolid + 0.01 μg/mL (1.0-1.0-1.0-1.0) Ofloxacin +Streptomycin Mefloquine + Moxifloxacin + 0.20 μg/mL (0.5-0.5-1.0-1.0)Ethionamide + Kanamycin

1.4 Determination of the Minimum Inhibitory Concentration (MIC)

The anti-tuberculosis activities were determined against M. tuberculosisin medium 7H9 and the MIC values were determined using the Alamar Blue(MABA) colorimetric method.

1.5 Determination of Synergism Between the Substances

The synergic interactions between the drugs tested were determinedthrough the Fractional Inhibitory Concentration Index (FIC) index, amethod widely accepted and used by the scientific community (Guidelinesof American Society for Microbiology). The calculations of the FIC weremade using the following formula:

Calculation of the FIC

(MIC of substance A, tested in combination)/MIC of substance A, testedalone+(MIC of substance B, tested in combination)/MIC of substance B,tested alone)

The interactions are evaluated as follows:

-   -   FIC≤0.5→synergic interactions    -   0.5<FIC≤4.0→additive interactions    -   FIC>4.0→antagonistic interactions

Example 1

Example 1 presents the results (Tables 1A and 1B) of the bestcombinations of the drug mefloquine with first choice drugs in thetreatment of tuberculosis.

TABLE 1A STRAIN H37RV - STANDARD STRAIN Combination MIC_(combination)FIC Proportion Mefloquine 12.5 μg/mL — — Mefloquine + 6.25 μg/mL 0.30.5-1.0 Pyrazinamide Mefloquine + Isoniazid 0.20 μg/mL 0.5 1.0-1.0

TABLE 1B STRAIN T3609 - RESISTANT TO OFLOXACIN AND STREPTOMYCINCombination MIC_(combination) FIC Proportion Mefloquine   25 μg/mL — —Mefloquine + Isoniazid 0.03 μg/mL 0.03 1.0-1.0

Example 2

Example 2 presents the results (Tables 2A and 2B) highlighting the bestcombinations between MFL and different second choice drugs, thefluoroquinolones and linezolid (LYN).

TABLE 2A STRAIN T3609 - RESISTANT TO OFLOXACIN AND STREPTOMYCINCombination MIC_(combination) FIC Proportion Mefloquine + Gatifloxacin0.62 μg/mL 0.5 1.0-0.5 Mefloquine + Moxifloxacin 1.25 μg/mL 0.5 1.0-0.5Mefloquine + Sparfloxacin 1.25 μg/mL 0.5 1.0-0.5

TABLE 2B STRAIN T113 - RESISTANT TO ISONIAZID. RIFAMPICIN. ETHAMBUTOLAND OFLOXACIN Combination MIC_(combination) FIC Proportion Mefloquine  25 μg/mL — — Mefloquine + Ofloxacin 1.25 μg/mL 0.5 1.0-0.5Mefloquine + Ciprofloxacin 0.62 μg/mL 0.5 1.0-0.5 Mefloquine +Levofloxacin 0.62 μg/mL 0.5 1.0-0.5

Example 3

The MICs of MFL, of each drug tested alone (MIC 1) and of each drugtested in combination (MIC 2), are expressed in Tables 3 and 4.

TABLE 3 COMBINATIONS OF MFL AND DIFFERENT FLUOROQUINOLONES. MFL + GATMFL + MOX MFL GAT MFL MOX Strain^(a) MIC 1 MIC 2 MIC 1 MIC 2 FIC MIC 1MIC 2 MIC 1 MIC 2 FIC H37Rv 12.5 0.12 0.12 0.12 1.0 12.5 0.15 0.25 0.150.6 T3609 12.5 0.31 0.62 0.31 0.5 12.5 0.62 1.25 0.62 0.5 T113 12.5 0.150.12 0.15 1.3 12.5 0.31 0.25 0.31 1.3 MFL + SPR MFL + OFX MFL SPR MFLOFX Strain^(a) MIC 1 MIC 2 MIC 1 MIC 2 FIC MIC 1 MIC 2 MIC 1 MIC 2 FICH37Rv 12.5 0.12 0.12 0.12 1.0 12.5 0.62 1.25 0.62 0.5 T3609 12.5 0.621.25 0.62 0.5 12.5 2.5 5.0 2.5 0.7 T113 12.5 0.12 0.12 0.12 1.0 12.50.62 1.25 0.62 0.5 MEF + CPX MEF + LVX MFL CPX MFL LVX Strain^(a) MIC 1MIC 2 MIC 1 MIC 2 FIC MIC 1 MIC 2 MIC 1 MIC 2 FIC H37Rv 12.5 0.62 0.620.62 1.0 12.5 0.62 0.62 0.62 1.0 T3609 12.5 3.12 5.00 3.12 0.9 12.5 2.502.50 2.50 1.2 T113 12.5 0.31 0.62 0.31 0.5 12.5 0.31 0.62 0.31 0.5 MIC1: MIC of the substance tested alone/MIC 2: MIC of the substance testedin combination. GAT—gatifloxacin, MOX—moxifloxacin, SPR—sparfloxacin,OFX—ofloxacin, CPX—ciprofloxacin, LVX—levofloxacin

TABLE 4 COMBINATIONS OF MFL AND LYN AND DIFFERENT DRUGS USED IN THEFIRST CHOICE TREATMENT OF TB. MFL + PYR MFL + ETB MFL PYR MFL ETBStrain^(a) MIC 1 MIC 2 MIC 1 MIC 2 FIC MIC 1 MIC 2 MIC 1 MIC 2 FIC H37Rv12.5 3.12 100 3.12 0.3 12.5 1.25 1.25 1.25 1.1 T3609 12.5 0.62 >100 —12.5 N.D N.D N.D. N.D. T113 12.5 0.62 >100 — 12.5 12.5  25.0  12.5  1.5MFL + INH MFL + LYN MFL INH MFL LYN Strain^(a) MIC 1 MIC 2 MIC 1 MIC 2FIC MIC 1 MIC 2 MIC 1 MIC 2 FIC H37Rv 12.5 0.10 0.20 0.10 0.5 12.5 0.620.62 0.62 1.0 T3609 12.5 0.015 0.5 0.015 0.03 12.5 N.D N.D N.D N.D T11312.5 3.12 6.25 3.12 0.7 12.5 0.31 0.50 0.31 0.6 MIC 1: MIC of thesubstance tested alone/MIC 2: MIC of the substance tested incombination. INH—isoniazid, PYR—pyrazinamide, ETB—ethambutol,LYN—linezolid N.D.—not determined

When combined with the first choice drugs, three synergic interactionswere observed: MFL+PYR and MFL+INH against the strain H37Rv and a strongsynergic interaction (FIC=0.03) between MFL and INH in relation tostrain T3609. INH and PYR play a fundamental role in the treatment ofTB. However, these drugs act in different phases of the development ofM. tuberculosis. INH has a bactericide effect on the bacteria that arein growth, while PYR possesses a sterilizing effect and acts on themicroorganisms that are latent.

In the light of the results hereby presented, it can be observed thatmefloquine presented the same MIC in relation to all the strains—thereis no cross-resistance. In addition to this, no antagonistic reaction ofthe mefloquine was observed in the combinations.

The pharmaceutical composition of this invention may be in any form thatis usually used to administer the drug for therapeutic purposes. Thus,the composition may be in the form of tablets, capsules, syrups, liquidsuspensions, elixirs, finely divided particles and similar substances.The pharmaceutical composition of this invention may include flavorings,colorings, and sweeteners or mixtures thereof.

The pharmaceutical composition of this invention may also includeexcipients selected from the group consisting of microcrystallinecellulose, lactose, crospovidone, corn starch, amino calcium alginate,poloxamer (polyoxyethylene-polyoxypropylene copolymer), talc, magnesiumstearate, sodium lauryl sulfate, calcium stearate, sodiumcarboxymethylcellulose, magnesium carbonate, carnauba wax, colophony,white beeswax, paraffin, sugar coating, acacia, gelatin, kaolin,titanium dioxide (E171), colloidal silicon dioxide, polyvinylpyrrolidoneK30, sucrose, Sunset Yellow (E110).

So the inventors demonstrated the synergic effect of the combination ofmefloquine with the first and second line drugs used in the treatment oftuberculosis and multi-drug resistant tuberculosis.

This invention is not limited to the materializations shown here, but isin accordance with a broad scope consistent with the principles and newaspects hereby described.

It should be understood that the examples and materializations herebydescribed are merely for illustrative purposes and that variousmodifications or alterations based thereon will occur to those skilledin the art and should be included within the claim scope.

REFERENCES

-   (1) World Malaria Report 2011, available at www.who.int/malaria.-   (2) Kunin, C. M.; Ellis, W. Y. Antimicrob Agents Chemother. 2000,    44(4); 848-852.-   (3) Tuberculosis (Edinb) 2002; 82(2-3); 85-90. Is Mycobacterium    tuberculosis a closer relative to Gram-positive or Gram-negative    bacterial pathogens? Fu L M; Fu-Liu C S.-   (4) Guidelines of American Society for Microbiology. Disponível em    www.aac.asm).

1. Pharmaceutical composition characterized by comprising: (a) 0.01 p/p of mefloquine; and, (b) 0.01 p/p of at least one, and a maximum of 3, drugs useful in the treatment of tuberculosis, selected from the group consisting of isoniazid, pyrazinamide, rifampicin, ethambutol, streptomycin, ethionamide, aminosalicylic acid, cycloserine, amikacin, kanamycin, capreomycin, linezolid, gatifloxacin, moxifloxacin, sparfloxacin, ofloxacin, ciprofloxacin and levofloxacin; and, (c) Optionally, excipients.
 2. Pharmaceutical composition in accordance with claim 1 characterized by the fact that the drug useful in the treatment of tuberculosis is preferably selected from at least one, and a maximum of 3, drugs of the group consisting of isoniazid, rifampicin, ethambutol, moxifloxacin and gatifloxacin.
 3. Pharmaceutical composition in accordance with claim 1 characterized by the fact that excipients are selected from the group consisting of microcrystalline cellulose, lactose, crospovidone, corn starch, amino calcium alginate, poloxamer (polyoxyethylene-polyoxypropylene copolymer), talc, magnesium stearate, sodium lauryl sulfate, calcium stearate, sodium carboxymethylcellulose, magnesium carbonate, carnauba wax, colophony, white beeswax, paraffin, sugar coating, acacia, gelatin, kaolin, titanium dioxide (E171), colloidal silicon dioxide, polyvinylpyrrolidone K30, sucrose, Sunset Yellow (E110).
 4. Use of mefloquine in a fixed dose in combination with at least one, and a maximum of 3, anti-tuberculosis drugs in the preparation of a medicament for the treatment of tuberculosis, where the anti-tuberculosis drugs are selected from the group isoniazid, pyrazinamide, rifampicin, ethambutol, streptomycin, ethionamide, aminosalicylic acid, cycloserine, amikacin, Kanamycin, capreomycin, linezolid, gatifloxacin, moxifloxacin, sparfloxacin, ofloxacin, ciprofloxacin and levofloxacin.
 5. Use of mefloquine in accordance with claim 4, where the anti-tuberculosis drugs are preferably selected from a least one, and a maximum of 3, drugs from the group consisting of isoniazid, rifampicin, ethambutol, moxifloxacin and gatifloxacin.
 6. Method for treating tuberculosis in a mammal that comprises administering, to said mammal in need of same, an effective quantity of mefloquine in combination with at least one, and a maximum of 3, anti-tuberculosis drugs, where the anti-tuberculosis drugs are selected from a group consisting of isoniazid, pyrazinamide, rifampicin, ethambutol, streptomycin, ethionamide, aminosalicylic acid, cycloserine, amikacin, Kanamycin, capreomycin, linezolid, gatifloxacin, moxifloxacin, sparfloxacin, ofloxacin, ciprofloxacin and levofloxacin. 